A DNA methylation-based test for efficiency of treatment is based on a plurality of genes. The test is suitable for monitoring treatment of subjects with neurological diseases, e.g., multiple sclerosis (MS); with cancer, e.g., ovarian and breast cancers, and with other diseases for which methylation of biomarkers differs in the diseased compared to the non-diseased state. The test is also suitable to differentiate non-affected from asymptomatic but affected and different states of diseases.
Several diagnostic tests are used to rule out or confirm cancer. For many cancers, the most definitive way to do this is to obtain a sample of the suspect tissue by biopsy followed by pathological analysis. However, biopsies are invasive, unpleasant procedures with their own associated risks, such as pain, bleeding, infection, and tissue or organ damage. In addition, if a biopsy does not result in an accurate or large enough sample, a false negative or misdiagnosis can result, often required that the biopsy be repeated. Monitoring treatment effects is also difficult.
Abnormal DNA methylation of cancer cells is reported. Tumor-specific changes in DNA methylation have been observed in many different malignancies and are frequently described as global hypomethylation combined with local hypermethylation. Global hypomethylation is linked to genomic instability of a tumor, whereas hypermethylation of specific genes correlates with their silencing and can induce point mutations owing to spontaneous deamination of 5me-C (transversion C→T). Silencing of a tumor suppressor gene can lead to enhanced transformation and increased tumor growth through disruption of the normal regulatory mechanisms of the affected cell.
Breast cancer, which is treatable by surgery, radiation therapy, chemotherapy, and hormonal therapy, is most often curable when detected in early stages. Mammography is the most important screening modality for the early detection of breast cancer. Breast cancer is classified into a variety of sub-types, but only a few of these affect prognosis or selection of therapy. Patient management following initial suspicion of breast cancer generally includes confirmation of the diagnosis, evaluation of stage of disease, and selection of therapy. Diagnosis may be confirmed by aspiration cytology, core needle biopsy with a stereotactic or ultrasound technique for nonpalpable lesions, or incisional or excisional biopsy. At the time the tumor tissue is surgically removed, part of it is processed for determination of Estrogen Receptor (ER) and Progesterone Receptor (PR) levels.
Prognosis and selection of therapy are influenced by the age of the patient, stage of the disease, pathologic characteristics of the primary tumor including the presence of tumor necrosis, estrogen-receptor (ER) and progesterone-receptor (PR) levels in the tumor tissue, and measures of proliferative capacity, as well as by menopausal status and general health. Overweight patients may have a poorer prognosis. Prognosis may also vary by race, with blacks, and to a lesser extent Hispanics, having a poorer prognosis than whites.
Three major treatments for breast cancer are surgery, radiation, and drug therapy. No treatment fits every patient, and often two or more treatments are required. The choice is determined by many factors, including the age of the patient and her menopausal status, the type of cancer (e.g., ductal vs. lobular), its stage, whether the tumor is hormone-receptive or not, and its level of invasiveness.
Breast cancer treatments are defined as local or systemic. Surgery and radiation are considered local therapies because they directly treat the tumor, breast, lymph nodes, or other specific regions. Drug treatment is called systemic therapy, because its effects are widespread. They may be used separately or, most often, in different combinations.
Ovarian cancer is the eighth most common cause of cancer mortality in women. It is diagnosed in more than 20,000 women in the USA each year and approximately 15,000 women die of the disease annually. Early detection and diagnosis of ovarian cancer has the potential to reduce cancer-related mortality and improve quality of life. Unfortunately, early stages of ovarian cancer are mostly asymptomatic, so its early detection has to rely on screening of ostensibly healthy population. This brings very high demands on the specificity and sensitivity of the assay, on the accessibility of patients' specimens, and on the overall cost of the procedure. In addition, compliance issues have to be considered—a painful, uncomfortable, or embarrassing procedure may be unpopular even if all the other components are in place. Tumor-specific DNA is present in the bloodstream and its methylation reflects the methylation in the primary tumor, while changes in methylation are linked to changes in gene expression and thus reflect long-term functional status of the tumor. Existing techniques are limited to imaging modalities such as transvaginal sonography (TVS) and blood-based biomarkers.
Other diseases for which methylation of DNA offers prognosis and treatment monitoring include neuroinflammatory demyelinating diseases, defined as any disease of the nervous system in which the myelin sheath of neurons is damaged and inflammation occurs, thereby impairing the conduction of signals in the affected nerves, causing impairment in sensation, movement, cognition, or other functions depending on which nerves are involved. The term describes the effect of the diseases, rather than their causes. Some demyelinating diseases are caused by infectious agents, some by autoimmune reactions, and some by unknown factors. Organo-phosphates, a class of chemicals that are the active ingredients in commercial insecticides such as sheep dip, weed-killers, and flea treatment preparations for pets and the like will also demyelinate nerves. Demyelinating diseases include multiple sclerosis (MS), acute disseminated encephalomyelitis, optic neuritis, transverse myelitis, Devic's disease, Guillain-Barre syndrome, chromic inflammatory demyelinating polyneuritis and progressive multifocal leukoencephalopathy (PML).
Multiple sclerosis (MS) is an example of a disease that is difficult to diagnose in its early stages. In fact, definite diagnosis of MS cannot be made until there is evidence of at least two anatomically separate demyelinating events occurring at least thirty days apart. The McDonald criteria represent international efforts to standardize the diagnosis of MS using clinical data, laboratory data, and radiologic data. Furthermore, a number of diseases can mimic the clinical presentation of MS, which requires an extensive prediagnosting evaluation.
There is an unmet need for accessible, inexpensive, and objective biomarkers for multiple sclerosis (MS) that can be used as a metric for the disease. Such biomarkers should distinguish MS after the initial clinically isolated syndrome and before new lesions develop in the CNS. They should also identify imminent exacerbations before clinical symptoms develop and additional CNS damage has already occurred. Ideally, such biomarkers should also identify exacerbations that do not produce clinical symptoms but nonetheless create CNS damage as evidenced by gadolinium-enhanced lesions.
Concentration of cfpDNA in blood of patients with RRMS is higher than in healthy controls (FIG. 1) and has specific methylation patterns, which can distinguish RRMS patients from healthy individuals, can separate patients who are undergoing clinical exacerbation [RRMS(E)] from those in remission [RRMS(R); Table 5 and distinguish patients treated with disease-modifying agents from those who are treatment naïve.
Disease exacerbation is defined, e.g., as presence of new or worsened symptoms and neurologic deficits lasting at least 72 hours.
Six drugs have been approved by the US Food and Drug Administration for treatment of MS as capable of decelerating its activity or progression, or both. All are parenteral, injected subcutaneously, intramuscularly or intravenously. Betaseron®, the first interferon beta (IFN-β) to be approved by the FDA (1993), was shown to reduce inflammation, decrease MS relapse rate, increase time between attacks, decrease the severity of attacks, and decrease the accumulated MS lesions as seen on MRIs of the brain. Betaseron® is administered every other day by subcutaneous injection, and is approved for relapsing forms of MS. Avonex® (IFN β-1a) was the first IFN-β to demonstrate and receive the FDA approval for slowing the rate of progression of disabilities in RR MS. It also decreases relapse rate and the accumulation of MS lesions on brain MRI. It is given as weekly intramuscular injections, and is indicated for the treatment of RRMS. Rebif® (IFN β-1a) although produced similarly to Avonex® by the Chinese hamster ovary cells, its formulation and administration are different to accommodate the thrice weekly subcutaneous route. Rebif® was proven efficacious in reducing the number and severity of relapses, delaying the progression of disabilities, and reducing the number of new and enlarging MS lesions on brain MRIs. It is approved for use in RRMS. Copaxone® (glatiramer acetate) was approved for decreasing the MS exacerbation rate and accumulation and activity of MS lesions on MRIs of the brain, albeit somewhat less than the interferons beta. It is administered daily by subcutaneous injection and is used in RRMS. Tysabri® (natalizumab) is the first monoclonal antibody approved by the FDA as monotherapy for relapsing forms of MS, and has shown efficacy in reducing relapse rates, progression of disabilities, and accumulation of new and active lesions on MRIs of the brain. Although head-to-head trials with IFNs β or glatiramer have not been done to validate comparison statements, numerically and statistically the efficacy of Tysabri® seemingly exceeds theirs, being in the 50% to 70% range clinically. It is administered intravenously once per month. A little over 25,000 MS patients are receiving it since marketing started 2 years ago. Novantrone® (mitoxantrone) is a cytotoxic agent that slows progression of disabilities and reduces the number of MS relapses. It is FDA approved for refractory SP and RR MS, and is recommended for use only over a limited time and in limited doses to avoid the serious adverse cardiac and myeloproliferative effects. It is administered intravenously, usually once every three months for up to two years. In addition, corticosteroids (prednisone, prednisolone, methylprednisolone, and dexamethasone) are used for the treatment of acute exacerbations to shorten their duration and speed up clinical recoveries. Although they have not been proven to alter the frequency of exacerbations or the progression of MS, the optic neuritis treatment trial has demonstrated that a single course of intravenous methylprednisolone (Solu-Medrol®)) was capable of reducing the accumulation of new lesions on brain MRIs over two years.
The practical use of medications to treat MS, however, faces important pitfalls. Treatment failure rate differs depending on criteria used to determine success. Their efficacy is usually modest or associated with significant side effects, and a large number of patients either do not respond to or discontinue them. Medication failure is usually determined clinically, after disease worsening, or appearance of new lesions on MRI imaging. Such determination takes significant amount of time (months or years) and sometimes after the appearance of irreversible neurological deficits. The clinical follow up of patients also includes monitoring of side effects that can range from flu-like symptoms to liver failure and death. At this point there are no means to predict patients' response to therapy and the potential scope of side effects. Thus, there is an unmeet need for objective laboratory-based biomarkers for treatment efficacy or related side effects that can be followed, and acted upon prior to the appearance of clinical symptoms.
Methylation-sensitive restriction enzyme digestion PCR (MSRE-PCR), can be used for rapid detection of DNA methylation in multiple fragments simultaneously. This procedure is based on extensive digestion of genomic DNA with methylation-sensitive restriction enzymes (MSRE) followed by multiplexed PCR amplification of user-defined genes using gene-specific primers. Although elimination of unmethylated fragments from the pool of potential PCR templates by MSRE digestion has been attempted previously, the requirements for high specificity and sensitivity of the assay present substantial problems that have been resolved in MSRE-PCR, which allows analysis of DNA methylation in a genomic equivalent of seven cells and can reliably detect methylation present in <2% of the sample.